Isolation And Culture Of Porcine Marrow Mesenchymal Stem Cells (pMSCs) And Developmental Potential Of Reconstructed Embryos By Nuclear Transfer

Two methods were compared for isolation and culture of porcine bone marrow mesenchymal stem cells (pMSCs), the growth characteristics of resulted pMSCs were determined and the develomental potential of these cells as somatic cell nuclear transfer(SCNT) donor cells were analysised;The developmental potential of human bone marrow mesenchymal stem cells (hMSCs) as human-pig interspecies somatic cell nuclear transfer (iSCNT) were anylysised too, the nuclei remodling pattern of these two types reconstructed embryos were compared. The results were as follows:1. Porcine fetal fibroblasts(PF) can be isolated more efficiently by 1mg/ml collagenase than 0.25% trypsinzation.and the isolated cells could achieve 70%~80% confluence in 3~4 days, So the primary porcine fibroblast cell line could be set up in shorter period.2. The numbers and purity of pMSCs derived from 1.077g/ml lymphocyte segregation liquid gradient centrifugation,are better than that of anchoring culture.3. PMSCs were identified by morphology and Cell markers. Cell markers CD106 and vimentin were positive , but CD45 was negative. Doubling time of pMSCs was 36.48h, the division index of cultured cells was 23.6±4.7‰in 4 day and the adhesive rate of cultured cells was 78.49±6.2% at 10h.4. Enucleate rate of porcine MII oocytes was 86.0±0.43% by the method of blinding aspiration.5. The cleavage rate(76.04±8.1% vs 74.45±12.1%)and the blastocyst rate (15.07±2.2% vs 14.63±7.9%)of the reconstructed embryos of pMSCs and PFs as donor cell showed without significant difference.Total cell numbers of blastocysts of the pMSCs reconstructed embryos was higher slightly than PFs(p>0.05).6. PMSCs and hMSCs as donor cells were transfered into porcine MII enucleate oocytes to produce reconstructed embryos,and the development capability of two types of reconstructed embryos was without significant difference before 72h;but the blastocyst rate of hMSCs-porcine inter-species reconstituted embryos was only 3.2±0.7% ,and the blastocyst rate of pMSCs reconstituted embryos was 11.59±2.35% (p<0.05).7. The morphology of nuclear stucture of hMSCs inter-species reconstituted embryos were observed after activition. hMSCs chromatin (39.39±10.6%) and pMSCs chromatin(53.49±9.5%) in were condensed stage at 2h after embryos activation (p>0.05). In addition, there were no significant differences in the formation of pronucleus between two types of embryos at 6h after activation.8. The numbers of hMSCs inter-species reconstituted embryos at differ stage were counted in 5d. The percentage of hMSCs inter-species reconstituted embryos at 4-cell stage was 28.80±3.9%,but there were only 13.04±1.1% and 14.06±1.5% in embryos of pMSCs as donor and PA embryos. (p<0.05).The results indicated that pMSCs have high proliferation capability and high consistency during passage,and can suport the reconstructed embryos developed to blastocyst in vitro. Comparing with the pMSCs reconstructed embryos, although hMSCs can be successfully remoldeling, the developmental potential of hMSCs reconstructed embryos is poor, most of them stop at 4~8-cell stage.